Fractionation and Centrifugation Flashcards
Tissues are blended and broken up by which piece of equipment?
Homogeniser.
Why are cells homogenised?
To release organelles from the cells so they can be studied.
What is the resultant fluid after homogenisation called?
Homogenate
Why is the homogenate filtered?
To remove large pieces of cell debris.
Why is the homogenate ice cold?
To slow down enzyme activity and prevent denaturing of enzymes.
What are the 2 reasons that the homogenate buffered?
To keep pH constant which ensures enzymes won’t denature and organelles maintain their structure.
Why is the homogenate isotonic?
Prevents water movement throughout the organelles which stops them from bursting or shrinking.
How must homogenate solutions be placed in the centrifuge?
Tubes must contain the same volume of homogenate and be placed opposite each other to ensure the centrifuge is balanced.
How are the largest organelles separated in a centrifuge?
What is this organelle?
The homogenate is spun at a slower speed which forced the denser organelles to the bottom of the tube, forming a pellet of nuclei.
What must the pellets produced be stored in and why?
Ice, to prevent changing of protein structure.
What is the fluid that remains in the test tube called?
Supernatant.
How are the next densest organelles separated?
What are they?
The supernatant is spun at an even higher speed, forming a pellet of mitochondria.
State the order of the next 3 densest organelles that separate.
Lysosomes
Golgi/ER
Ribosomes
